Phase-adaptive brain stimulation of striatal D1 medium spiny neurons in dopamine-depleted mice

Brain rhythms are strongly linked with behavior, and abnormal rhythms can signify pathophysiology. For instance, the basal ganglia exhibit a wide range of low-frequency oscillations during movement, but pathological “beta” rhythms at ~ 20 Hz have been observed in Parkinson’s disease (PD) and in PD animal models. All brain rhythms have a frequency, which describes how often they oscillate, and a phase, which describes the precise time that peaks and troughs of brain rhythms occur. Although frequency has been extensively studied, the relevance of phase is unknown, in part because it is difficult to causally manipulate the instantaneous phase of ongoing brain rhythms. Here, we developed a phase-adaptive, real-time, closed-loop algorithm to deliver optogenetic stimulation at a specific phase with millisecond latency. We combined this Phase-Adaptive Brain STimulation (PABST) approach with cell-type-specific optogenetic methods to stimulate basal ganglia networks in dopamine-depleted mice that model motor aspects of human PD. We focused on striatal medium spiny neurons expressing D1-type dopamine receptors because these neurons can facilitate movement. We report three main results. First, we found that our approach delivered PABST within system latencies of 13 ms. Second, we report that closed-loop stimulation powerfully influenced the spike-field coherence of local brain rhythms within the dorsal striatum. Finally, we found that both 4 Hz PABST and 20 Hz PABST improved movement speed, but we found differences between phase only with 4 Hz PABST. These data provide causal evidence that phase is relevant for brain stimulation, which will allow for more precise, targeted, and individualized brain stimulation. Our findings are applicable to a broad range of preclinical brain stimulation approaches and could also inform circuit-specific neuromodulation treatments for human brain disease.

Phase-adaptive brain stimulation (PABST). For PABST, we implanted 16-channel microwire optrodes (Microprobes) in the dorsal striatum (AP + 0.5/ML − 1.5/DV − 3.0; Fig. 1). We recorded LFPs across 16 channels using an Open Ephys system. The raw signal was amplified with a total gain of 198X, high-pass filtered at 0.1 Hz, and recorded with 16-bit resolution at a 30 k-Hz sampling rate.
To estimate phase, LFPs were streamed from the Open Ephys system to a notebook computer (Dell) via a zeroMQ data messaging system. Real-time analysis was performed in MATLAB; data were buffered over ~ 10 ms. Wavelet-based frequency and phase estimation were performed using a complex Morelet. Instantaneous frequency and phase were calculated using wavelets and predicted for 50 ms in the future using linear estimation.
During closed-loop experiments, D1DR+ mice with optical cannula were connected to the optical patch cable through a zirconia ferrule (Doric Lenses) without anesthesia. Light was generated using a 473-nm diodepumped solid-state (DPSS) laser source (OEM Laser Systems), and an optical rotary joint (Doric Lenses) was used to facilitate animal rotation. We used a 5 ms pulse width, which was controlled via TTL signals sent through a microcontroller controlled by a computer running PABST. Before each experiment, the power output of the laser was adjusted to ~ 10 milliWatts the fiber tip. Power measurements verified that the laser reached 90% power within 0.74 ms of TTL triggers and maintained ~ 10 milliWatts with < 5% error and stimulated spiking activity ( Figure S1). Of note, we delivered 4 Hz and 20 Hz PABST in the same animals with a randomized session order, making it possible to compare effects of PABST within mice with identical electrode locations and ChR2 expression.
Motion tracking. We captured motion using a 3-D motion-tracking system (OptiTrack); we have previously used this system to track mouse movement in detail 10,19 . Briefly, we implanted two 4-mm infrared-reflective spheres attached to the recording headstage in the anterior-posterior dimension. Four infrared cameras recorded the X (right-left), Y (forward-back), and Z (up-down) coordinates of the mouse's head at 120 frames/ per second (frames/s) to track head position 10 . Automated computer tracking data were synchronized with a video camera at 30 frames/s and neurophysiological recording hardware. mice were deeply anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and intracardially perfused with ice-cold 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde overnight and immersed in 30% sucrose until the brains sank. Brains were sectioned (40 or 50 μm) with a cryostat (Leica) and stored in PBS. Immunostaining procedures were performed with free-floating brain sections. Primary antibodies to tyrosine hydroxylase (rabbit anti-TH; Millipore −MAB152; 1:500-1000) were incubated overnight at 4 °C. Sections were visualized with Alexa Fluor fluorescent secondary antibodies goat anti-rat IgG Alexa 568, Thermo Fisher Scientific; 1:1000) and matched with the host primary by incubating 2 h at room temperature. Images were captured on an Olympus VS120 microscope.
Statistics. All procedures were reviewed by the Biostatistics, Epidemiology, and Research Design Core within the University of Iowa Institute for Clinical and Translational Science. To avoid making any distributional assumptions about the data, continuous measures were reported as medians and inter-quartile ranges. Testing for significant differences followed the non-parametric Wilcoxon rank sum test.

Results
We tested the hypothesis that the phase-adaptive brain stimulation (PABST) of dorsal striatal brain rhythms impacts behavior. We developed a real-time, closed-loop algorithm to calculate the ongoing instantaneous phase of brain rhythms from striatal LFPs to effect PABST. We streamed striatal LFPs to a dedicated computer for realtime preprocessing, filtering, and phase calculation in MATLAB. The sources of latency in these calculations were: (A) headstage to Open Ephys acquisition system latency: < 0.  1D). During optogenetic brain stimulation, the 473-nm laser achieved 90% power at 0.74 ms with 5 ms pulse width (Fig. 1E). Although it is difficult to simultaneously measure latencies and deliver stimulation, we estimated the total median latency as 12.98 ms. Because this latency is less than the 50 ms period of 20 Hz rhythms, these data indicate that this system latency is compatible with closed-loop PABST targeting dorsal striatal beta rhythms. We harnessed PABST to deliver closed-loop, real-time brain stimulation in the dorsal striatum. We depleted dopamine using the neurotoxin 6-OHDA, which decreased striatal dopaminergic terminals ( Fig. 2A,B). This manipulation reliably impairs movement and changes striatal oscillations 10,19,26 . We implanted recording electrodes in the mouse dorsomedial striatum (Fig. 2C) and virally-expressed ChR2+ in D1-MSNs (Fig. 2D). www.nature.com/scientificreports/ We estimated the phase-accuracy of PABST in targeting 4 Hz and 20 Hz dorsal striatal brain rhythms. Because all real-time brain stimulation systems have some lag, they must predict the phase at some point in the future, even if it is a few milliseconds (Fig. 3A). We estimated the phase of striatal LFPs (Fig. 3B-D) and delivered real-time, closed-loop stimulation pulses targeting 4 Hz and 20 Hz rhythms (Fig. 3E). We found that at 4 Hz the system produced an average phase difference of − 0.01 (− 0.90 to 0.82; Fig. 3F), and at 20 Hz the system produced an average phase difference of 0.10 (− 0.93 to 1.11; Fig. 3G). These data indicate that we can deliver phase-adaptive brain stimulation to striatal LFPs at 4 Hz and 20 Hz.
We tracked the position of mice at ~ 10-ms accuracy using an infrared tracking system (Fig. 4A). With this approach, we were able to record from striatal neuronal ensembles and LFPs in dopamine-depleted animals as they moved freely and were able to deliver real-time phase-responsive optogenetic stimulation of D1DR+ neurons (Fig. 4B). Of 358 recorded neurons, 73 had significantly increased firing rate with < 5 ms opto-stimulation latency ( Figure S1).
Next, we examined how PABST influenced striatal brain networks. We recorded LFPs and neuronal activity in six mice during 30-s epochs without stimulation with closed-loop stimulation targeting either 4 Hz or 20 Hz peaks. We found that there was no reliable difference in striatal power spectra between peak vs. trough stimulation ( Fig. 5A-F). However, we found consistent differences in spike-field coherence with 4 Hz and 20 Hz peak vs. trough stimulation (Spike-field coherence, 4 Hz peak: p = 0.005; Cohen's d = 6.2, Fig. 5G) and (Spike-field coherence, 20 Hz peak: p = 0.002; Cohen's d = 2.5, Fig. 5H). These data suggest that in the dorsal striatum, 4 Hz peak stimulation increased spike-field coherence, whereas 20 Hz peak stimulation decreased spike-field coherence.
Finally, we examined whether PABST influences head movement velocity, as has been shown previously for striatal D1-MSN stimulation 26,29 . We found that stimulation of D1-MSNs in-phase with 4 Hz oscillations at troughs, but not peaks, increased head movement speed (Fig. 6A). Of note, these movements were quite distinct from high-velocity dyskinetic movement observed with high-dose levodopa or with high-intensity optogenetics 10,30 (Supplemental Video 1). We compared velocity changes in six mice for peak vs. trough PABST stimulation relative to epochs with no stimulation (Fig. 6B,C). Strikingly, we found that 4 Hz trough PABST reliably increased velocity relative to 4 Fig. 6C]. However, we note that trough 4 Hz stimulation increased absolute head movement speed relative to no stimulation (p = 0.002; Cohen's d = 1.6; Figure S2), and both peak and trough 20 Hz stimulation increased head movement speed relative to no stimulation (peak 20 Hz; p = 0.002; Cohen's d = 3.3; trough 20 Hz; p = 0.002; Cohen's d = 1.7; Figure S2). Finally, we did not observe velocity improvements identical experiments with phase-random stimulation or in sham animals ( Figure S3). Taken together, these data suggest that the phase of 4 Hz PABST is relevant to D1-MSN stimulation of movement velocity. These data provide insight into the nature of D1-MSN firing relative to striatal brain rhythms and could be useful for future brain stimulation technologies.

Discussion
We interrogated striatal brain rhythm phase using adaptive brain stimulation. We used PABST to deliver optogenetic pulses at the peak phase or trough phase of ongoing striatal brain rhythms. First, we found that the latency of PABST was 13 ms, and that we could deliver phase-accurate stimulation at the peak/trough with 4 Hz and 20 Hz rhythms. Second, PABST targeting D1-MSNs powerfully modulated spike-field coherence of striatal brain rhythms, but not the overall power of striatal brain rhythms. Lastly, while both 4 Hz and 20 Hz improved head movement speed, 4 Hz trough PABST increased head movement velocity by 41% compared to 4 Hz peak PABST. These data directly test the hypothesis that the phase of ongoing brain rhythms is relevant to brain stimulation and provide insight into brain rhythms in the dorsal striatum. Neural systems are characterized by diverse and complex rhythms 1 . Our work demonstrates a novel, causal role for dorsal striatal brain rhythm phase in movements, albeit in a highly limited context-optogenetic D1-MSN stimulation of the dorsal striatum in dopamine-depleted mice. Our rationale for studying dopamine-depleted mice is that (1) they have readily detected deficits in movement that can be dynamically measured, and (2) dopamine-depletion markedly changes striatal brain rhythms which can be detected by phase-adaptive technologies such as PABST 10,19 . Notably, PABST had few reliable effects in sham-animals with intact dopaminergic circuits ( Figure S3). These data suggest that PABST may be effective under certain constraints, and future experiments with different behavioral measures and disease models will help clarify these constraints. However, our findings establish that PABST has the potential to be effective in rodent models of PD.
In the dorsal striatum, bursts of low-frequency rhythms can be powerfully affected by movement 13,31 . Because these rhythms are abnormal in basal ganglia disorders in preclinical models of movement disorders such as PD 10,11,15,17 , and because these rhythms synchronize striatal oscillations 32 , understanding the details of these oscillations is highly relevant for human disease. D1-MSN coherence with these oscillations was not explicitly related to movement, but 4 Hz trough PABST decreased D1-MSN coherence and improved head movement velocity. There may be specific populations of D1-MSNs that are linked with head movement and facilitated by specific spectral aspects of striatal LFPs. Future work will study these head-movement-specific D1-MSNs.
PABST is a new technology, combining off-the-shelf equipment to deliver phase-responsive stimulation with millisecond precision to dynamic striatal networks. This technology could be used, along with other similar www.nature.com/scientificreports/ techniques 33 to probe a variety of neuronal oscillations in the context of behavior and disease. Indeed, most preclinical and all clinical brain stimulation are delivered without consideration of these underlying brain rhythms and phase. Closed-loop technologies may be critical to adjusting brain stimulation to ongoing dynamics, so considering brain rhythm frequency and phase might help maximize the efficacy of brain stimulation for a wide range of applications 6,34 . We found that 4 Hz trough PABST of D1-MSNs is effective for improving head movement velocity. In the context of D1-MSNs, our work is consistent with prior work that has shown that D1-MSN stimulation facilitates movement 26,35 . Our work advances prior stimulation approaches by showing that highly precise 4 Hz trough PABST improved movement velocity in dopamine-depleted animals. It is possible that PABST can have complex effects on striatal networks affecting our results at 20 Hz. Understanding these effects could be important not only for a basic understanding of basal ganglia networks, but also for targeted brain stimulation interventions     20 Hz. However, we noticed that (G) 4 Hz peak-stimulation spike-field coherence was increased relative to trough spike-field coherence, whereas for (H) 20 Hz stimulation trough stimulation had a higher spike-field coherence than peak stimulation. Data from six dopamine-depleted mice.  8,36 . Head movement may not directly correlate with overall body movement, and there may many other movement effects of PABST that more advanced measurements of movement might capture 37 . Current adaptive brain stimulation approaches target the power in frequency-specific bands, whereas we target the phase 22 . While our work is currently a proof-of-concept in preclinical models, PABST has the potential to be more powerful while delivering fewer pulses, and thus more efficient, even when considering the computational requirements of PABST. Future work will investigate PABST's potential to treat movement disorders. D1-MSNs have been strongly implicated in dyskinesias 38 . Stimulation of these neurons with higher stimulation frequencies (10-20 Hz) has also been shown to promote dyskinesias 10,39 . We did not observe dyskinesias with stimulation in the present study, but dyskinesias may occur in animals receiving high-dose levodopa or brain stimulation. We have observed 4 Hz spike-field coherence around dyskinetic movements 10 . Because 4 Hz trough PABST can decrease spike-field coherence, it might be particularly effective in dyskinesias.
Our work has several limitations. First, all real-time, closed-loop systems involve finite computation time and must account for latency by predicting dynamic brain conditions in the future. Our phase-latency is 13 ms. It is possible that with higher fidelity models of future brain state, phase accuracy will improve, enabling more effective brain stimulation 40 . Second, it is striking that we did not observe differences in LFP power but did find differences in spike-field coherence. These data suggest that PABST affected local neuronal populations, but not wider brain networks. It is possible that with stimulation of more D1-MSNs, or another circuit element, we could achieve more powerful effects. Third, while we did not observe reliable effects of PABST in sham animals or with phase-random stimulation, it is possible that additional mice, control groups and PD models may facilitate further interpretation of stimulation effects. Fourth, we note that 20 Hz stimulation of ChR2 does not have 100% spike efficiency 6,41,42 and is far outside of the normal firing rate of D1-MSNs 43 . It is possible that other PABST strategies may have been more effective. Finally, our OptiTrack technology measures head movement, but there are many other aspects movements that our system does not capture 37,44 .
In summary, our work provides causal evidence of the relevance of phase to brain stimulation. Our phaseadaptive technology delivers low-latency stimulation which powerfully affects striatal spike-field coherence and increases movement velocity in dopamine-depleted mice. Future work will refine these methods and further refine PABST to deliver highly optimized and personalized brain stimulation with maximal efficacy.

Data availability
All code and raw data are available at https:// naray anan. lab. uiowa. edu. We stimulated D1-expressing medium spiny neurons (D1-MSNs) at the peak or trough of 4 Hz striatal brain rhythms using PABST; example trace from one mouse from an average epoch. (B) We found that 4 Hz trough PABST increased head movement velocity compared to 4 Hz peak stimulation, suggesting that 4 Hz phase may be relevant to brain stimulation. (C) By contrast, the phase of 20 Hz rhythms did not reliably affect movement velocity. Data from six mice.